Neurotransmitter regulating active peptide, purification method and application of neurotransmitter regulating active peptide in sleep improving products

Abstract

The invention belongs to the technical field of active peptides, and particularly relates to a neurotransmitter regulating active peptide, a purification method thereof and application of the neurotransmitter regulating active peptide in sleep improving products. The invention provides a neurotransmitter regulating active peptide derived from cowhide, which can improve secretion of GABA and 5-HT in nerve cells and reduce DA expression, also shows good sedative and hypnotic effects in in-vivo experiments, and is expected to be applied to sleep improvement products.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to a neurotransmitter-regulating bioactive peptide, its purification method, and its application in sleep improvement products. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Sleep-promoting peptides have attracted widespread attention in scientific research and application in recent years. Due to their natural biological functions, low side effects, and good therapeutic effects, these peptides are becoming potential candidates to replace traditional hypnotic drugs. Many studies have focused on elucidating the molecular mechanisms by which peptides improve sleep, primarily examining how they regulate brain activity to achieve a hypnotic effect by interacting with specific neurotransmitter pathways (such as the GABA system and circadian rhythm pathways). For example, Δsleep-inducing peptides bind to multiple neurotransmitter receptors to form complexes that exert multiple sleep-enhancing effects.

[0004] Peptides have attracted attention in recent years as potential sleep promoters. These natural peptides, due to their low toxicity, fewer side effects, and significant biological activity, show promising applications in addressing insomnia and sleep problems in sub-healthy populations. For example, Wang, J. et al. isolated sleep-promoting peptides (PBPs) from fish bones, and identified that the amino acid sequence TG7 exhibited the best sleep-promoting activity.

[0005] In view of the current state of research, this invention believes that collagen peptides, which have good biocompatibility and are already widely used in various functional foods with good safety, can be used to develop sleep-aiding active peptides with higher economic value, which will help enhance the commercial value of collagen peptides. Summary of the Invention

[0006] In a first aspect, the present invention provides a neurotransmitter-regulating active peptide, wherein the preparation method of the active peptide includes the following steps: Collagenase is added to crude bovine collagen protein for enzymatic hydrolysis, and the portion of the hydrolysate with a molecular weight less than 3kDa is retained as the active peptide; the collagenase is a product of Bacillus subtilis or Bacillus coagulans under gelatin-induced fermentation.

[0007] The first aspect mentioned above also includes the following preferred technical solutions: The crude bovine collagen protein is prepared as follows: after defatting the bovine hide, it is crushed and extracted with acid to obtain crude bovine collagen protein.

[0008] Furthermore, the degreasing process involves soaking the hide in an isopropanol solution with a concentration of 8-12% for 20-40 hours. Before degreasing, the fat and fascia tissue on the surface of the hide are thoroughly removed, and the hide is cut into small pieces before being soaked for degreasing.

[0009] Furthermore, the acid solution is preferably an inorganic acid solution, specifically including but not limited to formic acid, acetic acid, or propionic acid, with a concentration of 500-700 mmol / L, an extraction time of 70-80 h, and the extraction process is carried out at room temperature.

[0010] The collagenase is prepared as follows: Bacillus subtilis or Bacillus coagulans is inoculated into a seed culture medium and fermented for a period of time, then inoculated into a fermenter for further cultivation. The inoculation amount is 5-7%, the stirring speed is 220-270 r / min, the aeration rate is 2.5-3.5 L / min, the fermentation temperature is 30-38℃, the fermentation time is 30-36 h, and the fermentation medium contains 0.4-0.6% wt gelatin. The collagenase is obtained by salting out the fermentation product.

[0011] Furthermore, the salting-out process involves precipitation with ammonium sulfate, and the saturation of ammonium sulfate is 50-60%. Compared to the previous method, this method changes the amount of ammonium sulfate added, thereby screening and enriching the collagenases and improving the enzymatic hydrolysis efficiency of crude bovine collagen protein.

[0012] The enzymatic hydrolysis is performed with the following parameters: collagenase is added to crude bovine collagen protein for enzymatic hydrolysis at an enzyme dosage of 1200-1700 U / g crude protein; the reaction time is 1.5-3 h; the temperature is 36-40℃; and the pH is 7.0-7.2. After the enzymatic hydrolysis is completed, the reaction is terminated by high-temperature inactivation. The reaction system is then centrifuged, and the solution portion is retained. Ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The retained portion is the active peptide.

[0013] Furthermore, to further obtain bioactive peptides with small molecular weight and good water solubility, this invention also designs a method for purifying the above-mentioned bioactive peptides by salting out. Verification has shown that the purified peptides obtained have a more significant sleep-promoting effect compared to the above-mentioned bioactive peptides. Therefore, in a second aspect, this invention provides a method for purifying the bioactive peptides described in the first aspect, comprising the following steps: (1) Place the active peptide described in the first aspect in an ice bath, add ammonium sulfate to the saturation of 10-20%, stir for 20-40 min, centrifuge and retain the supernatant; (2) Add ammonium sulfate to the supernatant obtained in step (1) until the saturation is 40-70%, then centrifuge and retain the precipitate; (3) The precipitate from step (3) is desalted by dialysis to obtain the purified peptide.

[0014] The main purpose of step (1) above is to remove small molecule impurities such as miscellaneous proteins and polymeric peptides. In a better implementation, the active peptide is placed in an ice bath and stirred continuously at a speed of 80~120 r / min; a suitable centrifugation speed is 7000~9000 r / min for 15~25 min.

[0015] The purpose of step (2) above is to enrich the target peptide. The appropriate ammonium sulfate saturation is 40-70%. The polypeptide corresponding to the above salt saturation has high sleep-aiding activity. In a better embodiment, the ammonium sulfate saturation is 50-60%, and even more so, 60%.

[0016] In step (3) above, the molecular weight cutoff for dialysis is 1 kDa.

[0017] In a third aspect, the present invention provides a pharmaceutical composition comprising the active peptide described in the first aspect, or a purified peptide prepared by the purification method described in the second aspect.

[0018] In a fourth aspect, the present invention provides the use of the neurotransmitter-regulating active peptide described in the first aspect and the pharmaceutical composition described in the third aspect in the preparation of a sleep-improving product.

[0019] The aforementioned sleep-improving products are oral preparations, and further, are drugs, health products, or dietary supplements. In one embodiment of the present invention with better efficacy, the above-mentioned active peptides or pharmaceutical compositions are used to prepare related health products, and further, are gels, tablets, fractions, capsules, or powders.

[0020] Compared with the prior art, the beneficial effects of the present invention are: In the inventors' prior research, a polypeptide product with ACE-inhibiting activity was provided, which was obtained by enzymatic hydrolysis of crude fish skin collagen protein by enzymes secreted by Bacillus subtilis or Bacillus coagulans under gelatin induction. In subsequent research, this invention found that the bovine collagen polypeptide prepared by the above method exhibited good neurotransmitter regulatory biological activity, and could regulate the secretion of neurotransmitters such as γ-aminobutyric acid (GABA), 5-hydroxytryptophan (5-HT), and dopamine (DA) in brain nerve cells, which helps to achieve sedation and hypnosis in organisms. Furthermore, the above polypeptide can penetrate the blood-brain barrier and act on the hypothalamus to regulate neurotransmitters in the hypothalamus of animals, effectively shortening the sleep latency and prolonging sleep time. Attached Figure Description

[0021] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0022] Figure 1 The effect of the polypeptide product described in Example 4 on the 5-HT content in cell supernatant; Figure 2 The effect of the polypeptide product described in Example 4 on the DA content in the cell supernatant; Figure 3 The effect of the polypeptide product described in Example 4 on the GABA content in the cell supernatant. Detailed Implementation

[0023] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0024] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0025] In the context of this specification, the word "including" is considered to mean "particularly including". It should not be interpreted as "consisting of only".

[0026] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0027] Example 1 This embodiment provides a method for extracting active peptides from bovine hide. The active peptides have a molecular weight of less than 3 kDa and have been verified to have good sedative and sleep-aiding activities. The extraction method steps are as follows: (1) Bacillus coagulans was inoculated into seed culture medium (1% beef extract, 2% peptone, 0.5% NaCl, balance water, pH 7.5), fermented at 38℃, and cultured in shake flasks for 24 h. Then it was transferred to a fermenter (2.0% glucose, 0.5% peptone, 0.4% gelatin, 0.1% CaCl2, 0.4% Na2HPO4, 0.03% KH2PO4, balance water, pH 7.5), with an inoculum size of 6%, a stirring speed of 250 r / min, an aeration rate of 3 L / min, a fermentation temperature of 30℃, and a fermentation time of 36 h. After fermentation, the fermentation broth was centrifuged at 6500 r / min and 4℃ for 15 min, and the supernatant was retained. While stirring, add the supernatant to 60% saturation, let stand at 4°C for 4 hours, centrifuge and discard the precipitate; continue adding ammonium sulfate to the supernatant to 80% saturation, let stand at 4°C overnight, centrifuge and collect the precipitate, retain the precipitate portion and transfer it to 0.2 mol / L pH 7.8 phosphate buffer for reconstitution, and dialyze with water 1-3 times to obtain collagenase for later use.

[0028] (2) After washing the cowhide clean, cut it into small pieces and soak it in 10% isopropanol solution for defatting. The soaking time is 20-40h. After that, rinse it clean and freeze dry it. Then put it into a planetary ball mill and grind it to a particle diameter of 200μm. Weigh the ground cowhide powder and add it to 10 times the volume of acetic acid solution (600mmol / L). Stir continuously for 72h. Centrifuge at 12000r / min for 10min and retain the supernatant. Add NaCl to precipitate and centrifuge to retain the precipitate to obtain crude cowhide collagen protein.

[0029] (3) Add the collagenase obtained in step (1) to the crude bovine collagen protein from step (2), with an addition amount of 1500 U / g, pH of 7.2, a hydrolysis time of 2 h, and a hydrolysis temperature of 38 °C. After the reaction is complete, heat the reaction system to 100 °C for 10 min to terminate the reaction. Then, centrifuge the hydrolysate and discard the precipitate. Use a 3 kDa ultrafiltration membrane to ultrafilter the supernatant after centrifugation, and retain the portion with a molecular weight less than 3 kDa, which is the active peptide.

[0030] Example 2 In this embodiment, another polypeptide is provided. The difference from Example 1 is that the active peptide obtained in step 3 of Example 1 is further purified by salting out. The specific steps are as follows: (1) Place the above active peptide in an ice bath and stir slowly (100 r / min). Add ammonium sulfate powder in batches until the saturation reaches 20%. Keep stirring in the ice bath for 30 min. Then centrifuge at 4°C and 8000 r / min for 20 min. Discard the precipitate and keep the supernatant.

[0031] (2) Take the supernatant and continue stirring. Add ammonium sulfate powder in batches to increase the saturation to 60% so that the target peptide can be fully precipitated. Then centrifuge at 4℃ and 10000r / min for 25min and retain the precipitate.

[0032] (3) Add the precipitate obtained in step (2) to 10 mL of 0.05 mol / L pH 7.2 phosphate buffer, stir magnetically to dissolve, and obtain a concentrated peptide solution. Transfer the solution into a dialysis bag with a molecular weight cutoff of 1 kDa, seal it, and place it in a beaker containing 500 mL of sterile deionized water. Dialyze at 4°C, changing the deionized water every 4 hours for a total of 3 dialysis cycles. Stir gently each time the water is changed to improve the desalination efficiency.

[0033] After dialysis, the peptide solution was removed and centrifuged at 4°C and 8000 r / min for 10 min to remove a small amount of insoluble impurities, resulting in desalted purified peptides.

[0034] Comparative Example 1 In this embodiment, a bovine hide polypeptide is provided, and the preparation method of the extract is as follows: Add a compound enzyme preparation to the crude bovine collagen protein obtained in step (2) of Example 1. The compound enzyme is a mixture of papain and collagenase at a dosage ratio of 1:1, with an addition amount of 1500 U / g. The enzymatic hydrolysis time is 2 h, the enzymatic hydrolysis temperature is 38 °C, and the enzymatic hydrolysis reaction is terminated by heating to 100 °C for 10 min. Then, the enzymatic hydrolysate is centrifuged, and the supernatant is passed through a 3 kDa ultrafiltration membrane. The portion with a molecular weight less than 3 kDa is retained to obtain the bovine peptide.

[0035] Example 4 In this embodiment, the bioactivity of the polypeptide products obtained in Examples 1-2 and Comparative Example 1 was verified, including in vitro activity verification and in vivo activity verification in mice. The research methods are as follows: I. In vitro activity verification (a) Test materials Cell lines: Mouse hypothalamic neuron cell line (GT1-7), used to determine the effect of peptides on GABA; human neuroblastoma cell line (SH-SY5Y), used to determine the effect of peptides on 5-HT and DA.

[0036] Polypeptide samples: the active peptide obtained in Example 1, the purified peptide obtained in Example 2, and the bovine skin polypeptide obtained in Comparative Example 1 (purity ≥95%, prepared into a 10 mmol / L stock solution with PBS and stored at -20℃).

[0037] Reagents required for detection: fetal bovine serum, serum-free DMEM medium, MTT assay kit, 5-HT / GABA / DA ELISA kit, trypsin, PBS buffer.

[0038] (II) Research Methods 1. Determining Dosage Concentration using the MTT Assay SH-SY5Y cells in the logarithmic growth phase were harvested at a concentration of 5 × 10⁻⁶. 3 Cells were seeded per well in a 96-well plate and incubated in DMEM medium containing 10% fetal bovine serum for 24 h at 37°C and 5% CO2. Gradient concentrations of peptide solutions (5, 10, 25, 50, 100, 200 μmol / L) were added. GABA (100 μmol / L) was added to the positive control group, and an equal volume of serum-free medium was added to the blank control group. After culturing for another 24 h, an MTT assay was performed. Based on cell viability ≥85%, the low and high doses of the three peptides were confirmed to be 10 and 100 μmol / L, respectively.

[0039] 2. Detection of sleep-related neurotransmitter secretion using ELISA method SH-SY5Y and GT1-7 cells were harvested at a concentration of 2 × 10⁻⁶. 5 Cells were seeded per well in a 24-well plate and cultured for 24 hours. Then, the culture medium was replaced with serum-free medium and starved for 12 hours. The corresponding concentration of peptide solution was added and cultured for another 24 hours. The supernatant of cells from each well was collected, centrifuged at 10,000 r / min and 4℃ for 10 min, and the supernatant was used for later use.

[0040] Take the supernatant of SH-SY5Y cell culture and follow the instructions of the ELISA kit for 5-HT and DA. Set up a standard curve and measure the absorbance at the corresponding wavelengths (5-HT: 450nm; DA: 450nm) using an ELISA reader. Calculate the concentration of the two neurotransmitters in the supernatant.

[0041] Take the supernatant of GT1-7 cell culture and use a GABA ELISA kit to detect it. Measure the absorbance at 450 nm using an ELISA reader and calculate the GABA concentration.

[0042] 3. Evaluation Results The active peptide obtained in Example 1 is designated as peptide A1, the purified peptide obtained in Example 2 is designated as peptide A2, and the bovine hide polypeptide obtained in Comparative Example 1 is designated as peptide D1. The effects of the above three polypeptides on the contents of 5-HT, DA, and GABA in SH-SY5Y and GT1-7 cells are as follows: Figure 1-3 As shown, the GABA group could not exclude the added GABA in the supernatant, so the positive group was not included in the statistics.

[0043] according to Figure 1-3 The results showed that the addition of A1 peptide and A2 peptide to the above-mentioned polypeptide components significantly increased the secretion of 5-HT and GABA (P<0.05), while reducing the expression of DA, proving that A1 peptide and A2 peptide have a regulatory effect on neurotransmitter components in brain cells and help enhance the sedative and hypnotic effect.

[0044] Meanwhile, the bovine skin polypeptide provided in Comparative Example 1 had a partial regulatory effect on the above-mentioned neurotransmitters, but the effect was not significant.

[0045] II. In vivo activity verification in mice (a) Experimental Consumables Experimental animals: SPF grade ICR mice, male, weighing 20-22g, 12 mice per group, acclimatized for 3 days (temperature 22±2℃, humidity 50±5%, 12h light / 12h dark cycle).

[0046] Experimental reagents: A1 peptide and A2 peptide (dissolved in physiological saline), diazepam (1 mg / kg, positive control, dissolved in physiological saline), physiological saline (blank control), mouse brain tissue homogenate kit, 5-HT / GABA / DA ELISA kit, sodium pentobarbital; The experimental animals were divided into a blank control group, a control group, and an experimental group, and the administration methods were as follows: Control group: administered normal saline by gavage; Control group: Diazepam solution (1 mg / kg) administered by gavage; Experimental group: Low / high dose group: Gavage administration of polypeptide solution (40 mg / kg, 80 mg / kg, based on in vitro concentration conversion).

[0047] (II) Research Methods 1. Sodium pentobarbital synergistic sleep experiment Thirty minutes after the last administration, all mice were intraperitoneally injected with a subhypnotic dose of sodium pentobarbital (50 mg / kg). Observe and record the sleep latency (time from injection to the disappearance of the righting reflex) and sleep duration (time from the disappearance to the recovery of the righting reflex); record three times for each mouse and take the average value.

[0048] 2. Detection of neurotransmitter content in brain tissue After sleep monitoring, mice were euthanized by cervical dislocation, the brain was quickly dissected, and hypothalamic tissue was harvested. After weighing, pre-cooled physiological saline was added at a ratio of 1:10, and the mixture was homogenized on ice. The homogenate was centrifuged at 4°C and 12,000 r / min for 15 min, and the supernatant was used as the brain tissue extract. The contents of 5-HT, GABA, and DA in the extract were detected using an ELISA kit, following the same procedure as in in vitro experiments. The concentration of neurotransmitters per unit weight of brain tissue (ng / mg) was calculated.

[0049] 3. Test Indicators During the feeding and experimental process, the mice maintained normal body weight and food intake, and there were no significant differences in liver and kidney function indicators. Their behavioral indicators and neurotransmitter changes are shown in Table 1 below: Table 1. Changes in behavioral indicators and neurotransmitters in mice. According to the results shown in Table 1, the active peptides obtained in Examples 1 and 2 can significantly shorten the sleep latency in mice (P<0.01) and significantly prolong sleep time (P<0.01), and the high-dose group of A2 peptide has activity close to that of positive drugs.

[0050] The levels of GABA and 5-HT in the hypothalamus were significantly increased (P<0.05), while the level of DA was significantly decreased (P<0.05), showing a dose-dependent effect. The effect of A2 peptide was better than that of A1 peptide and was close to that of the positive control drug diazepam.

[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A neurotransmitter-regulating active peptide, characterized in that, The preparation method of the active peptide includes the following steps: After defatting, the cowhide is crushed and extracted with acid to obtain crude cowhide collagen protein. Collagenase is added to the crude cowhide collagen protein for enzymatic hydrolysis. The portion of the enzymatic hydrolysis product with a molecular weight of less than 3kDa is the active peptide. The collagenase is a product of Bacillus subtilis or Bacillus coagulans under gelatin-induced fermentation. The collagenase is prepared as follows: Bacillus subtilis or Bacillus coagulans is inoculated into a seed culture medium and fermented for a period of time, then inoculated into a fermenter for further cultivation. The inoculation amount is 5-7%, the stirring speed is 220-270 r / min, the aeration rate is 2.5-3.5 L / min, the fermentation temperature is 30-38℃, the fermentation time is 30-36 h, and the fermentation medium contains 0.4-0.6% wt gelatin. The collagenase is obtained by salting out the fermentation product.

2. The neurotransmitter-regulating active peptide as described in claim 1, characterized in that, The salting out is achieved by adding ammonium sulfate to precipitate the salt, and the saturation of ammonium sulfate is 50-60%.

3. The purification method of the neurotransmitter-regulating active peptide according to any one of claims 1-2, characterized in that, Includes the following steps: (1) Place the active peptide in an ice bath, add ammonium sulfate to a saturation of 10-20%, stir for 20-40 min, centrifuge and retain the supernatant; (2) Add ammonium sulfate to the supernatant obtained in step (1) until the saturation is 40-70%, then centrifuge and retain the precipitate; (3) The precipitate from step (3) is desalted by dialysis to obtain the purified peptide.

4. The purification method for neurotransmitter-regulating active peptides as described in claim 3, characterized in that, In step (1), the active peptide is placed in an ice bath and stirred continuously at a speed of 80~120 r / min; centrifugation is performed at a speed of 7000~9000 r / min for 15~25 min.

5. The purification method for neurotransmitter-regulating active peptides as described in claim 3, characterized in that, In step (2), the ammonium sulfate saturation is 40-70%, and further, it is 60%.

6. The purification method for neurotransmitter-regulating active peptides as described in claim 3, characterized in that, In step (3), the molecular weight cutoff for dialysis is 1 kDa.

7. A pharmaceutical composition, characterized in that, The composition includes the active peptide according to claim 1 or 2, or the purified peptide prepared by the purification method according to any one of claims 3-6.

8. The use of the neurotransmitter-regulating active peptide of claim 1 or 2, or the pharmaceutical composition of claim 7, in the preparation of a sleep-improving product.

9. The application as described in claim 8, characterized in that, The sleep-improving product is an oral preparation, and further, a drug, health product, or dietary supplement.

10. The application as described in claim 9, characterized in that, The active peptide or pharmaceutical composition is a health product, and more specifically, a gel, tablet, granule, capsule, or powder.

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